Abstract

Although cellular processes depend on protein-protein interactions, our understanding of molecular recognition between proteins remains far from comprehensive. Protein-protein interfaces are structural and energetic mosaics in which a subset of interfacial residues, called hot spots, contributes disproportionately to the affinity of the complex. These hot-spot residues can be further clustered into hot regions. It has been proposed that binding energetics between residues within a hot region are cooperative, whereas those between hot regions are strictly additive. If this idea held true for all protein-protein interactions, then energetically significant long-range conformational effects would be unlikely to occur. In the present study, we show cooperative binding energetics between distinct hot regions that are separated by >20 A. Using combinatorial mutagenesis and surface plasmon resonance binding analysis to dissect additivity and cooperativity in a complex formed between a variable domain of a T cell receptor and a bacterial superantigen, we find that combinations of mutations from each of two hot regions exhibited significant cooperative energetics. Their connecting sequence is composed primarily of a single beta-strand of the T cell receptor variable Ig domain, which has been observed to undergo a strand-switching event and does not form an integral part of the stabilizing core of this Ig domain. We propose that these cooperative effects are propagated through a dynamic structural network. Cooperativity between hot regions has significant implications for the prediction and inhibition of protein-protein interactions.

Equilibrium binding analysis of single-site variants. The changes in free energy for each of the single-site hVβ2.1 mutants binding to TSST-1 are plotted. The dotted red line indicates the threshold value used to distinguish energetically significant versus insignificant mutations.

Two hot regions for TSST-1 interaction in hVβ2.1. (A) Ribbon diagram/molecular surface representation of the hVβ2.1 domain. The molecule is color-coded according to hot regions (CDR2 hot region, red; FR3 hot region, blue) and the c″ β-strand that connects the two hot regions (green). (B) Model of the hVβ2.1–TSST-1 complex. The hVβ2.1 CDR2 and FR3 hot regions are color-coded as in A; TSST-1 is yellow. The hVβ2.1 CDR2 (red) and FR3 (blue) hot regions are shown interfacing the additional TSST-1 hot region (purple) and the TSST-1 hot region originally described (orange) (), respectively.

A dynamic structural network for the propagation of cooperative binding effects. (A and B) Strand swapping of the c″ β-strand in TCR Vβ domains as depicted in the hVβ2.1 domain (A) () and the mVβ2.3 domain (B) (). The strands that are hydrogen-bonded to one another are colored orange. (C) A view of the hVβ2.1 domain in which the protein core and the CDR2 (red) and FR3 (blue) hot regions and the connecting c″ β-strand (green) are outlined by ovals on the left and right, respectively. (D–G) The c′–c″ or c″–d β-strand regions of hVβ2.1 from a TCR-superantigen structure [Protein Data Bank (PDB) ID code 1KTK] (D), hVβ2.1 from a TCR-autoimmune peptide-MHC structure (PDB ID code 1YMM) (E), mVβ2.3 (PDB ID code 1KB5) (F), and mVβ8.2 (PDB ID code 1BEC) (G).